br In this study we aimed to o
In this study, we aimed to oﬀer a comprehensive comparison be-tween the protein coronas that formed in vivo onto clinically used li-posomes in healthy and tumor-bearing mice, by LC-MS/MS. Our results, provided the first evidence that protein corona fingerprinting indeed varies, both quantitatively (Fig. 1C) and qualitatively (Figs. 2B and 4B), in the absence and presence of tumor growth. These diﬀerences ob-served between the protein coronas formed in melanoma and human lung carcinoma models compared to healthy control mice, supported the hypothesis that the interaction of liposomes with blood-circulating proteins is greatly aﬀected by the ongoing pathophysiological events.
High abundance, high MW proteins, such as albumin and im-munoglobulins present in the blood circulation hinder the detection of the low MW blood proteomic fractions, likely to contain previously unidentified disease-specific biomarkers. In addition, the rapid clear-ance of small proteins from the blood circulation by kidney Oxidopamine hydrochloride is another reason why the low MW region of the blood proteome remains largely unexplored. The only way a small molecule can remain in the blood circulation for longer periods is to adhere to a long-circulating, high abundance protein, such as albumin. Plasma depletion meth-odologies, often used as a pre-fractionation tool to address the issue of the ‘large dynamic concentration range’ of plasma proteome, discard those carrier proteins and inevitably their valuable cargos .
Our data in this study suggest that the liposomal corona results in an ‘enriched’ sampling of the blood proteome, by minimising the ‘noise’ from highly abundant proteins, contrary to plasma control (Fig. 1D). It should be emphasized that albumin was not depleted from plasma control or corona samples. Proteomic analysis of the recovered in vivo corona resulted in a significantly increased number of identified pro-teins in comparison to conventional proteomic analysis of plasma samples (Fig. 2A). Even though albumin and other highly abundant plasma proteins were found to interact with the surface of liposomes (in both healthy and tumor-bearing mice) the extensive purification pro-cesses applied to retrieve the corona-coated liposomes and purify them from the unbound proteins, worked as fractionation tool and increased the range of plasma protein detection (Fig. 2A and B). Interestingly, classification of the corona proteins according to their
Fig. 5. Candidate protein biomarkers, with diﬀerential abundance in the liposomal coronas formed in healthy and lung carcinoma-bearing SCID nude mice: (A) Only proteins with at least 2 fold change with the lowest p value are shown. The full list of proteins with p < 0.05 are shown in Table S9. Data was filtered to a 1% false discovery rate (FDR); (B) Ingenuity Pathway Analysis (IPA) of potential biomarker corona proteins associated with lung carcinoma. Upregulated proteins (red; n = 21) and downregulated proteins (green; n = 22) are organised according to their cellular localisation (extracellular, plasma membrane, cyto-plasmic, nuclear). The name of proteins illustrated in the diagram and their respective gene symbol, p values and fold change values are shown in Table S10; (C) List with human proteins (secreted from A549-luc cells) identified in the coronas of intravenously injected liposomes in lung-carcinoma bearing mice. Only proteins with two or more human-specific peptides are shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
M. Hadjidemetriou et al.
molecular weight demonstrated that the liposomal surface was mainly covered by low MW proteins (Figs. 2C and 4C). It is possible that the low MW proteins identified have high aﬃnity and interact directly with the surface of PEGylated liposomes (also known as ‘hard’ corona pro-teins) and/or they are trapped between other corona-carrier proteins that are adhered to the NPs surface (also known as ‘soft’ corona pro-teins) . It should be emphasized, that the manner in which proteins adsorb onto the NPs surface is highly dependent on the NPs physico-chemical properties including their size, surface curvature and func-tionalization. However, concrete relationships between nanomaterials synthetic identity and protein corona composition in complex biolo-gical fluids remain vague . Diﬀerent proteins could be either en-riched or weakly bound depending on their aﬃnity for the NP surface, which is defined by the balance between the rates of association (Kon) and dissociation (Koﬀ). The stability of the liposomal formulation em-ployed in this study after intravenous administration, the preferential capturing of low MW plasma proteins and the eﬀective recovery and purification of liposomes from the blood circulation played an im-portant role in the uncovering of low abundant disease-specific pro-teins. Whether diﬀerent nanomaterials could allow the detection of distinct biomarker signatures remains to be investigated in future stu-dies.